Food Biochemistry and Food Processing (2 edition)

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138 Part 2: Biotechnology and Enzymology

model, whose principles are (i) there is a thermodynamic equi-

librium between the transition state and the state of reactants at

the top of the energy barrier and (ii) the rate of chemical reac-

tion is proportional to the concentration of the particles in the

high-energy transition state.

Combining basic kinetic expressions, which describe a simple

chemical reaction:

A + B C

Scheme 7.6.

and thermodynamic equations, such as (Finkelstein and Ptitsyn

2002)

G=−RTlnk

G=H−TS

Eyring’s equation is formed:

k=

kβT

h

e−(

GRT)

=

kβT

h

e−(

HRT−SR)

where k is the reaction velocity, R is the universal gas

constant (8.3145 J·mol−^1 K),kB the Boltzmann’s constant

(1.381· 10 −^23 J·K−^1 ),his the Plank constant (6.626· 10 −^34 J·s)

andT is the absolute temperature in degrees Kelvin (K). If

we compare Eyring’s equation and Arrhenius equation (Laidler

1984), we can realise thatEandHare parallel quantities, as we

concluded above. Eyring’s equation is an appropriate and useful

tool, which allows us to simplify the complicated meaning of

thermodynamics and, simultaneously, to interpret experimental

data to physical meanings.

Enzyme Dynamics During Catalysis

Multiple conformational changes and intramolecular motions

appear to be a general feature of enzymes (Agarwal et al. 2002).

The structures of proteins and other biomolecules are largely

maintained by non-covalent forces and are therefore subject to

thermal fluctuations ranging from local atomic displacements to

complete unfolding. These changes are intimately connected to

enzymatic catalysis and are believed to fulfil a number of roles in

catalysis: enhanced binding of substrate, correct orientation of

catalytic groups, removal of water from the active site and trap-

ping of intermediates. Enzyme conformational changes may be

classified into four types (Gutteridge and Thornton 2004): (i)

domain motion, where two rigid domains, joined by a flexi-

ble hinge, move relative to each other; (ii) loop motion, where

flexible surface loops (2–10 residues) adopt different conforma-

tions; (iii) side chain rotation, which alters the position of the

functional atoms of the side chain and (iv) secondary structure

changes.

Intramolecular motions in biomolecules are usually very fast

(picosecond–nanosecond) local fluctuations. The flexibility as-

sociated with such motions provides entropic stabilisation of

conformational states (Agarwal et al. 2002). In addition, there are

also slower- (microsecond–millisecond) and larger-scale, ther-

mally activated, transitions. Large-scale conformational changes

are usually key events in enzyme regulation.

ENZYME PRODUCTION

In the past, enzymes were isolated primarily from natural

sources, and thus a relatively limited number of enzymes were

available to the industry (Aehle 2007). For example, of the hun-

dred or so enzymes being used industrially, over one half are

from fungi and yeast and over a third are from bacteria, with the

remainder divided between animal (8%) and plant (4%) sources

(Panke and Wubbolts 2002, van Beilen and Li 2002, Bornscheuer

2005). Today, with the recent advances of molecular biology and

genetic engineering, several expression systems were developed

and exploited and used for the commercial production of several

therapeutic (Mcgrath 2005), analytical or industrial enzymes

(van Beilen and Li 2002, Kirk et al. 2002, Otero and Nielsen

2010). These systems have not only improved the efficiency,

availability and cost with which enzymes can be produced, but

they have also improved their quality (Labrou and Rigden 2001,

Andreadeli et al. 2008, Kapoli et al. 2008, Kotzia and Labrou

2009, Labrou 2010).

Enzyme Heterologous Expression

There are two basic steps involved in the assembly of every

heterologous expression system:

1. The introduction of the DNA, encoding the gene of inter-
   est, into the host cells, which requires: (i) the identification
   and isolation of the gene of the protein we wish to express,
   (ii) insertion of the gene into a suitable expression vector
   and (iii) introduction of the expression vector into the se-
   lected cell system that will accommodate the heterologous
   protein.


2. The optimisation of protein expression by taking into
   account the effect of various factors such as growing
   medium, temperature, and induction period.

A variety of vectors able to carry the DNA into the host

cells are available, ranging from plasmids, cosmids, phagemids,

viruses as well as artificial chromosomes of bacterial, yeast or

human origin (BAC, YAC or HAC, respectively; Grimes and

Monaco 2005, Monaco and Moralli 2006, Takahashi and Ueda

2010). The vectors are either integrated into the host chromoso-

mal DNA or remain in an episomal form. In general, expression

vectors have the following characteristics (Fig. 7.11):

1. Polylinker: it contains multiple restriction sites that facili-
   tate the insertion of the desired gene into the vector.


2. Selection marker: encodes for a selectable marker, allow-
   ing the vector to be maintained within the host cell under
   conditions of selective pressure (i.e. antibiotic).


3. Ori: a sequence that allows for the autonomous replication
   of the vector within the cells.


4. Promoter: inducible or constitutive, that regulates RNA
   transcription of the gene of interest.